A family of UDP-galactose; β-N-acetyl-glucosamine β1-3galactosyltransferases (β3Gal-T's) was recently identified (Amado, M., Almeida, R., Carneiro, F., et al. A family of human β3-galactosyltransferases: characterisation of four members of a UDP-galactose β-N-acetylglucosamine/β-N-acetylgalactosamine β1,3-Galactosyltransferase family. J. Biol. Chem. 273:12770-12778, 1998; Kolbinger, F., Streiff, M. B. and Katopodis, A. G. Cloning of a human UDP-galactose:2-acetamido-2-deoxy-D-glucose 3β-galactosyltransferase catalysing the formation of type 1 chains. J. Biol. Chem. 273:433-440, 1998; Hennett, T., Dinter, A., Kuhnert, P., Mattu, T. S., Rudd, P. M. and Berger, E. G. Genomic cloning and expression of three murine UDP-galactose: β-N-acetylglucosamine β1,3-galactosyltransferase genes. J. Biol. Chem. 273:58-65, 1998; Miyaki, H., Fukumoto, S., Okada, M., Hasegawa, T. and Furukawa, K. Expression cloning of rat cDNA encoding UDP-galactose G(D2) β1,3 galactosyltransferase that determines the expression of G(D1b)/G(M 1)G(A1). J. Biol. Chem. 272:24794-24799, 1997). Three genes within this family, β3Gal-T1, -T2, and -T3, encode β3galactosyltransferases that form the Galβ1-3GlcNAc linkage. The type 1 chain Galβ1-3GlcNAc sequence is found in both N- and O-linked oligosaccharides of glycoproteins and in lactoseries glycosphingolipids, where it is the counterpart of type 2 Galβ1-4GlcNAc poly-N-acetyllactosamine structures (Kobata. A. Structures and functions of the sugar chains of glycoproteins. Eur J Biochem 209:483-501, 1992.). Type 1 chain structures are found mainly in endodermally derived epithelia, whereas the type 2 chains are found in ecto- and mesodermally derived cells including erythrocytes (Oriol, R., Le Pendu, J. and Mollicone, R. Genetics of ABO, H, Lewis, X and related antigens. Vox Sanguinis 51:161-171, 1986; Clausen, H. and Hakomori, S. ABH and related histo-blood group antigens; immunochemical differences in carrier isotypes and their distribution. Vox Sanguinis 56:1-20, 1989). Normal gastro-intestinal epithelia express mainly type 1 chain glycoconjugates, while type 2 chain structures are predominantly expressed in tumors (Hakomori, S. Aberrant glycosylation in tumors and tumor-associated carbohydrate antigens. Tumor malignancy defined by aberrant glycosylation and sphingo(glyco)lipid metabolism. Advances in Cancer Research 52:257-331, 1989; Hakomori, S. Tumor malignancy defined by aberrant glycosylation and sphingo(glyco)lipid metabolism. Cancer Res 56:5309-5318, 1996). It is of considerable interest to define the gene(s) responsible for formation of these core structures in normal and malignant epithelia. Several characteristics of the three previously described β3Gal-Ts capable of forming type 1 chain structures suggest that these are not the major enzyme(s) involved in type 1 chains synthesis in epithelia: (i) Northern analysis indicates that β3Gal-T1 and -T2 are exclusively expressed in brain (Amado, M., Almeida, R., Carneiro, F., et al. family of human β3-galactosyltransferases: characterisation of four members of a UDP-galactose β-N-acetylglucosamine/β-N-acetylgalactosamine β1,3-Galactosyltransferase family. J. Biol. Chem. 273:12770-12778, 1998; Kolbinger, F., Streiff, M. B. and Ktopodis, A. G. Cloning of a human UDP-galactose:2-acetamido-2-deoxy-D-glucose β3-galactosyltransferase catalysing the formation of type 1 chains. J. Biol. Chem. 273:433-440, 1998; Hennett, T., Dinter, A., Kuhnert, P., Mattu, T. S., Rudd, P. M. and Berger, E. G. Genomic cloning and expression of three murine UDP-galactose: β-N-acetylglucosamine β1,3-galactosyltransferase genes. J. Biol. Chem. 273:58-65, 1998); (ii) although β3Gal-T3 has a wider expression pattern it is not detected in several tissues including colon and it is weakly expressed in gastric mucosa (Amado, M., Almeida, R., Carneiro, F., et al. A family of human β3-galactosyltransferases: characterisation of four members of a UDP-galactose β-N-acetylglucosamine/β-N-acetylgalactosamine β1,3-Galactosyltransferase family. J. Biol. Chem. 273:12770-12778, 1998; Kolbinger, F., Streiff, M. B. and Ktopodis, A. G. Cloning of a human UDP-galactose:2-acetamido-2-deoxy-D-glucose β3-galactosyltransferase catalysing the formation of type 1 chains. J. Biol. Chem. 273:433-440, 1998); (iii) the kinetic properties of recombinant enzymes are not consistent with those reported for βGal-T activities in epithelia (Sheares, B. T., Lau, J. T. and Carlson, D. M. Biosynthesis of galactosyl-beta 1,3-N-acetylglucosamine. J. Biol. Chem. 257:599-602, 1982; Holmes, E. H. Characterization and membrane organization of beta 1-3 and beta 1-4 galactosyltransferases from human colonic adenocarcinoma cell lines Cob 205 and SW403: basis for preferential synthesis of type 1 chain lacto-series carbohydrate structures. Arch Biochem Biophys 270:630-646, 1989); and (iv) the acceptor substrate specificities of β3Gal-T1, -T2, or -T3 do not include the mucin-type core 3 structure (Amado, M., Almeida, R., Carneiro, F., et al. A family of human β3-galactosyltransferases: characterisation of four members of a UDP-galactose β-N-acetylglucosamine/β-N-acetylgalactosamine β1,3-Galactosyltransferase family. J. Biol. Chem. 273:12770-12778, 1998; Hennett, T., Dinter, A., Kuhnert, P., Mattu, T. S., Rudd, P. M. and Berger, E. G. Genomic cloning and expression of three murine UDP-galactose: β3-N-acetylglucosamine β1,3-galactosyltransferase genes. J. Biol. Chem. 273:58-65, 1998), which was previously found to be a highly efficient substrate for β3Gal-T activity isolated from porcine trachea (Sheares, B. T. and Carlson, D. M. Characterization of UDP-galactose:2-acetamido-2-deoxy-D-glucose 3 beta-galactosyltransferase from pig trachea. J. Biol. Chem. 258:9893-9898, 1983).
Access to additional existing βGlcNAc β3Gal-transferase genes encoding β3Gal-transferases with better kinetic properties than β3Gal-T1, -T2, and -T3 would allow production of more efficient enzymes for use in galactosylation of oligosaccharides, glycoproteins, and glycosphingolipids. Such enzymes could be used, for example, in pharmaceutical or other commercial applications that require synthetic galactosylation of these or other substrates that are not or poorly acted upon by β3Gal-T1, -T2, and -T3, in order to produce appropriately glycosylated glycoconjugates having particular enzymatic, immunogenic, or other biological and/or physical properties.
Consequently, there exists a need in the art for additional isolated UDP-galactose: β-N-acetyl-glucosamine β1-3Galactosyltransferases having unique, specific properties and the primary structure of the genes encoding these enzymes. The present invention meets this need, and further presents other related advantages, as described in detail below.